The Na/K-ATPase enzyme is ubiquitously expressed in most eukaryotic cells and is essential for maintaining the trans-membrane ion gradient by pumping Na+ out and K+ into cells. Structurally, the enzyme consists of two non-covalently linked α and β subunits Similar to other P-ATPases, including the gastric H/K-ATPase and sarcoplasmic reticulum Ca-ATPase (SERCA), the Na/K-ATPase α subunit has 10 transmembrane domains with both N- and C-termini located in the cytoplasm. Based on the published crystal structures of both Na/K-ATPase and SERCA, the α subunit consists of several well-characterized domains: actuator (A) domain consists of the N-terminus and the second cytosolic domain (CD2) connected to transmembrane helices M2 and M3; highly conserved discontinuous phosphorylation (P) domain is close to the plasma membrane; and a relatively isolated nucleotide-binding (N) domain. These structures also show a significant movement of A and N domain during the ion pumping cycle. It appears that the A domain rotates while the N domain closes up during the transport cycle, which opens (E1) and closes (E2) the A, N and P domains. Interestingly, these domains have also been implicated in interacting with many protein partners, including inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), phosphoinositide 3′ kinase (PI3K), phospholipase C-γ (PLC-γ), ankyrin, and cofilin.
Previously, the inventors and others have demonstrated that binding of cardiotonic steroids (CTS) such as ouabain to the Na/K-ATPase stimulates multiple protein kinase cascades. Moreover, knockout of Src abolishes most of these activations. Src, a member of Src family non-receptor kinases, plays an important role in the signal transduction pathways of many extracellular stimuli, i.e., cytokines, growth factors and stress responses and has been considered as a promising target for therapeutic interventions in certain cancers and bone diseases. Several endogenous inhibitors of Src have been documented previously, including c-terminal Src kinase (CSK), CSK-homologous kinase (CHK), Wiscott-Aldrich syndrome protein (WASP), RACK1 and caveolin.
The Na/K-ATPase interacts directly with Src via at least two binding motifs: one being between the CD2 of the α1 subunit and Src SH2; and, other involving the third cytosolic domain (CD3) and Src kinase domain. The formation of this Na/K-ATPase and Src complex serves as a receptor for ouabain to provoke protein kinase cascades. Specifically, binding of ouabain to Na/K-ATPase will disrupt the latter interaction, and then result in assembly and activation of different pathways including ERK cascades, PLC/PKC pathway and mitochondrial ROS production. Moreover, this interaction keeps Src in an inactive state. Thus, the Na/K-ATPase functions as an endogenous negative Src regulator. This is consistent with the fact that the basal Src activity is inversely correlated to the amount of Na/K-ATPase α1 subunit in both cultured cells and in α1 heterozygous mouse tissues. See also, the co-inventors' pending application PCT/US07/023,011, filed Oct. 17, 2007 (Pub. No. WO 2808/054792 on May 8, 2008), claiming priority from U.S. Ser. No. 60/855,482 filed Oct. 16, 2006, which are expressly incorporated herein by reference.
There is still a need to determine the molecular interaction between the Na/K-ATPase and Src in order to then develop novel Src modulators that may be used to antagonize ouabain-induced signal transduction.
Moreover, there is a need for targeting the newly discovered Na/K-ATPase/Src receptor complex to develop novel agonists or antagonists of the receptor so that the receptor function of Na/K-ATPase/Src complex can be either stimulated for treating diseases such as ischemia/reperfusion injury or inhibited for treating diseases such as tissue fibrosis, congestive heart failure, and cancer.
Such a general method would be of tremendous utility in that whole families of related proteins each with its own version of the functional domain of interest could be identified. Knowledge of such related proteins would contribute greatly to our understanding of various physiological processes, including cell growth or death, malignancy, renal/cardiovascular function and immune reactions.
Such a method would also contribute to the development of increasingly more effective therapeutic, diagnostic, or prophylactic agents having fewer side effects.
According to the present invention, just such novel compositions and methods are provided.